Use of microparticles combined with submicron oil-in-water emulsions

ABSTRACT

Compositions are provided which include biodegradable microparticles with entrapped or adsorbed antigens, in combination with submicron oil-in-water emulsions. Also provided are methods of immunization which comprise administering to a vertebrate subject (a) a submicron oil-in-water emulsion, and (b) a therapeutically effective amount of a selected antigen entrapped in a microparticle.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/935,466 filed Aug. 20, 2001, now U.S. Pat. No. 6,458,370, which is acontinuation of U.S. patent application Ser. No. 09/564,416, filed May2, 2000, now U.S. Pat. No. 6,306,405, which is a divisional of U.S.patent application Ser. No. 09/015,736, filed Jan. 29, 1998, now U.S.Pat. No. 6,086,901, from which applications priority is claimed pursuantto 35 U.S.C. §120 and this application is related to Provisional PatentApplication Ser. No. 60/069,724, filed Dec. 16, 1997, from whichpriority is claimed under 35 U.S.C. §1 19(e)(1), and which applicationsare incorporated herein by reference in their entireties.

TECHNICAL FIELD

The present invention relates generally to vaccine compositions. Inparticular, the invention relates to the use of biodegradablemicroparticles including entrapped or adsorbed antigens, in combinationwith submicron oil-in-water emulsions.

BACKGROUND OF THE INVENTION

Numerous vaccine formulations which include attenuated pathogens orsubunit protein antigens, have been developed. Conventional vaccinecompositions often include immunological adjuvants to enhance immuneresponses. For example, depot adjuvants are frequently used which adsorband/or precipitate administered antigens and which can retain theantigen at the injection site. Typical depot adjuvants include aluminumcompounds and water-in-oil emulsions. However, depot adjuvants, althoughincreasing antigenicity, often provoke severe persistent localreactions, such as granulomas, abscesses and scarring, when injectedsubcutaneously or intramuscularly. Other adjuvants, such aslipopolysacharrides, can elicit pyrogenic responses upon injectionand/or Reiter's symptoms (influenza-like symptoms, generalized jointdiscomfort and sometimes anterior uveitis, arthritis and urethritis).Saponins, such as Quillaja saponaria, have also been used asimmunological adjuvants in vaccine compositions against a variety ofdiseases.

More particularly, Complete Freund's adjuvant (CFA) is a powerfulimmunostimulatory agent that has been successfully used with manyantigens on an experimental basis. CFA includes three components: amineral oil, an emulsifying agent, and killed mycobacteria, such asMycobacterium tuberculosis. Aqueous antigen solutions are mixed withthese components to create a water-in-oil emulsion. Although effectiveas an adjuvant, CFA causes severe side effects primarily due to thepresence of the mycobacterial component, including pain, abscessformation and fever. CFA, therefore, is not used in human and veterinaryvaccines.

Incomplete Freund's adjuvant (IFA) is similar to CFA but does notinclude the bacterial component. IFA, while not approved for use in theUnited States, has been used elsewhere in human vaccines for influenzaand polio and in veterinary vaccines for rabies, canine distemper andfoot-and-mouth disease. However, evidence indicates that both the oiland emulsifier used in IFA can cause tumors in mice.

Muramyl dipeptide (MDP) has been found to be the minimal unit of themycobacterial cell wall complex that generates the adjuvant activityobserved with CFA. See, e.g., Ellouz et al., Biochem. Biophys. Res.Commun. (1974) 59:1317. Several synthetic analogs of MDP have beengenerated that exhibit a wide range of adjuvant potency and sideeffects. For a review of these analogs, see, Chedid et al., Prog.Allergy (1978) 25:63. Representative analogs of MDP include threonylderivatives of MDP (Byars et al., Vaccine (1987) 5:223), n-butylderivatives of MDP (Chedid et al., Infect. Immun. 35:417), and alipophilic derivative of a muramyl tripeptide (Gisler et al., inInmmunomodulations of Microbial Products and Related Synthetic Compounds(1981) Y. Yamamura and S. Kotani, eds., Excerpta Medica, Amsterdam, p.167).

One lipophilic derivative of MDP isN-acetylmuramyl-L-alaflyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero3-hydroxyphosphoryloxy-ethylamine(MTP-PE). This muramyl tripeptide includes phospholipid tails that allowassociation of the hydrophobic portion of the molecule with a lipidenvironment while the muramyl peptide portion associates with theaqueous environment. Thus, the MTP-PE itself is able to act as anemulsifying agent to generate stable oil-in-water emulsions. MTP-PE hasbeen used in an emulsion of 4% squalene with 0.008% TWEEN 80®, termedMTP-PE-LO (low oil), to deliver the herpes simplex virus gD antigen witheffective results (Sanchez-Pescador et al., J. Immunol. (1988)141:1720-1727), albeit poor physical stability. Recently, MF59, a safe,highly immunogenic, submicron oil-in-water emulsion which contains 4-5%w/v squalene, 0.5% w/v TWEEN 80®, 0.5% SPAN 85®, and optionally, varyingamounts of MTP-PE, has been developed for use in vaccine compositions.See, e.g., Ott et al., “MF59—Design and Evaluation of a Safe and PotentAdjuvant for Human Vaccines” in Vaccine Design: The Subunit and AdjuvantApproach (Powell, M. F. and Newman, M. J. eds.) Plenum Press, New York,1995, pp. 277-296.

Despite the presence of such adjuvants, conventional vaccines often failto provide adequate protection against the targeted pathogen. In thisregard, there is growing evidence that vaccination against intracellularpathogens, such as a number of viruses, should target both the cellularand humoral arms of the immune system.

More particularly, cytotoxic T-lymphocytes (CTLs) play an important rolein cell-mediated immune defense against intracellular pathogens such asviruses and tumor-specific antigens produced by malignant cells. CTLsmediate cytotoxicity of virally infected cells by recognizing viraldeterminants in conjunction with class I MHC molecules displayed by theinfected cells. Cytoplasmic expression of proteins is a prerequisite forclass I MHC processing and presentation of antigenic peptides to CTLs.However, immunization with killed or attenuated viruses often fails toproduce the CTLs necessary to curb intracellular infection. Furthermore,conventional vaccination techniques against viruses displaying markedgenetic heterogeneity and/or rapid mutation rates that facilitateselection of immune escape variants, such as HIV or influenza, areproblematic. Accordingly, alternative techniques for vaccination havebeen developed.

Particulate carriers with adsorbed or entrapped antigens have been usedin an attempt to elicit adequate immune responses. Such carriers presentmultiple copies of a selected antigen to the immune system and promotetrapping and retention of antigens in local lymph nodes. The particlescan be phagocytosed by macrophages and can enhance antigen presentationthrough cytokine release. Examples of particulate carriers include thosederived from polymethyl methacrylate polymers, as well as microparticlesderived from poly(lactides) and poly(lactide-co-glycolides), known asPLG. Polymethyl methacrylate polymers are nondegradable while PLGparticles biodegrade by random nonenzymatic hydrolysis of ester bonds tolactic and glycolic acids which are excreted along normal metabolicpathways.

Recent studies have shown that PLG microparticles with entrappedantigens are able to elicit cell-mediated immunity. For example,microencapsulated human immunodeficiency virus (HIV) gp120 has beenshown to induce HIV-specific CD4+ and CD8+ T-cell responses in mice(Moore et al., Vaccine (1995) 13:1741-1749). Similarly,microparticle-encapsulated ovalbumin has been shown to be capable ofpriming cellular immune responses in vivo and can induce mucosal IgAresponses when administered orally (O'Hagan et al., Vaccine (1993)11:149-154). Additionally, both antibody and T-cell responses have beeninduced in mice vaccinated with a PLG-entrapped Mycobacteriumtuberculosis antigen (Vordermeier et al., Vaccine (1995) 13:1576-1582).Antigen-specific CTL responses have also been induced in mice using amicroencapsulated short synthetic peptide from the circumsporozoiteprotein of Plasmodium berghei.

However, the use of microparticles with entrapped or adsorbed antigen,in combination with submicron oil-in-water emulsions, has not heretoforebeen described.

DISCLOSURE OF THE INVENTION

The present invention is based on the surprising and unexpecteddiscovery that the use of biodegradable microparticles, such as thosederived from a poly((α-hydroxy acid), and including entrapped oradsorbed antigen, in combination with submicron oil-in-water emulsions,serves to enhance the immunogenicity of the antigen. The use of suchcombinations provides a safe and effective approach for enhancing theimmunogenicity of a wide variety of antigens.

Accordingly, in one embodiment, the invention is directed to acomposition comprising a submicron oil-in-water emulsion, and a selectedantigen entrapped in, or adsorbed to, a biodegradable microparticle.

In another embodiment, the invention is directed to a compositioncomprising (a) a submicron oil-in-water emulsion which comprises 4-5%w/v squalene, 0.25-0.5% w/v TWEEN 80®, and 0.5% w/v SPAN 85®, andoptionally,N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy-ethylamine,and (b) a selected antigen entrapped in, or adsorbed to, a biodegradablemicroparticle.

In yet another embodiment, the subject invention is directed to a methodof immunization which comprises administering to a vertebrate subject(a) a submicron oil-in-water emulsion, and (b) a therapeuticallyeffective amount of a selected antigen entrapped in, or adsorbed to, abiodegradable microparticle.

In still further embodiments, the invention is directed to a method ofmaking a composition comprising combining a submicron oil-in-wateremulsion with a selected antigen entrapped in, or adsorbed to, abiodegradable microparticle.

In particularly preferred embodiments, the microparticle is derived froma poly(α-hydroxy acid), preferably poly(L-lactide), poly(D,L-lactide) orpoly(D,L-lactide-co-glycolide).

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows total IgG titers at 2, 6 and 10 weeks following initialvaccination in mice immunized with gp120; gp120+MF59; PLG with entrappedgp120; and PLG with entrapped gp120+MF59.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds.,Academic Press, Inc.); and Handbook of Experimental Immunology, Vols.I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell ScientificPublications); and Sambrook, et al., Molecular Cloning: A LaboratoryManual (2nd Edition, 1989).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

I. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

The term “microparticle” as used herein, refers to a particle of about100 nm to about 150 μm in diameter, more preferably about 200 nm toabout 30 μm in diameter, and most preferably about 500 nm to about 10 μmin diameter. Preferably, the microparticle will be of a diameter thatpermits parenteral administration without occluding needles andcapillaries. Microparticle size is readily determined by techniques wellknown in the art, such as photon correlation spectroscopy, laserdiffractometry and/or scanning electron microscopy. Microparticles foruse herein will be formed from materials that are sterilizable,non-toxic and biodegradable. Such materials include, without limitation,poly(α-hydroxy acid), polyhydroxybutyric acid, polycaprolactone,polyorthoester, polyanhydride. Preferably, microparticles for use withthe present invention are derived from a poly(α-hydroxy acid), inparticular, from a poly(lactide) (“PLA”) or a copolymer of D,L-lactideand glycolide or glycolic acid, such as a poly(D,L-lactide-co-glycolide)(“PLG” or “PLGA”), or a copolymer of D,L-lactide and caprolactone. Themicroparticles may be derived from any of various polymeric startingmaterials which have a variety of molecular weights and, in the case ofthe copolymers such as PLG, a variety of lactide:glycolide ratios, theselection of which will be largely a matter of choice, depending in parton the coadministered antigen. These parameters are discussed more fullybelow.

By “antigen” is meant a molecule which contains one or more epitopesthat will stimulate a host's immune system to make a cellularantigen-specific immune response when the antigen is presented, or ahumoral antibody response. Normally, an epitope will include betweenabout 3-15, generally about 5-15, amino acids. For purposes of thepresent invention, antigens can be derived from any of several knownviruses, bacteria, parasites and fungi. The term also intends any of thevarious tumor antigens. Furthermore, for purposes of the presentinvention, an “antigen” refers to a protein which includesmodifications, such as deletions, additions and substitutions (generallyconservative in nature), to the native sequence, so long as the proteinmaintains the ability to elicit an immunological response. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe antigens.

An “immunological response” to an antigen or composition is thedevelopment in a subject of a humoral and/or a cellular immune responseto molecules present in the composition of interest. For purposes of thepresent invention, a “humoral immune response” refers to an immuneresponse mediated by antibody molecules, while a “cellular immuneresponse” is one mediated by T-lymphocytes and/or other white bloodcells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (“CTL”s). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote theintracellular destruction of intracellular microbes, or the lysis ofcells infected with such microbes. Another aspect of cellular immunityinvolves an antigen-specific response by helper T-cells. Helper T-cellsact to help stimulate the function, and focus the activity of,nonspecific effector cells against cells displaying peptide antigens inassociation with MHC molecules on their surface. A “cellular immuneresponse” also refers to the production of cytokines, chemokines andother such molecules produced by activated T-cells and/or other whiteblood cells, including those derived from CD4+ and CD8+ T-cells.

A composition or vaccine that elicits a cellular immune response mayserve to sensitize a vertebrate subject by the presentation of antigenin association with MHC molecules at the cell surface. The cell-mediatedimmune response is directed at, or near, cells presenting antigen attheir surface. In addition, antigen-specific T-lymphocytes can begenerated to allow for the future protection of an immunized host.

The ability of a particular antigen or composition to stimulate acell-mediated immunological response may be determined by a number ofassays, such as by lymphoproliferation (lymphocyte activation) assays,CTL cytotoxic cell assays, or by assaying for T-lymphocytes specific forthe antigen in a sensitized subject. Such assays are well known in theart. See, e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doeet al., Eur. J. Immunol. (1994) 24:2369-2376; and the examples below.

Thus, an immunological response as used herein may be one whichstimulates the production of CTLs, and/or the production or activationof helper T-cells. The antigen of interest may also elicit anantibody-mediated immune response. Hence, an immunological response mayinclude one or more of the following effects: the production ofantibodies by B-cells; and/or the activation of suppressor T-cellsand/or γδ T-cells directed specifically to an antigen or antigenspresent in the composition or vaccine of interest. These responses mayserve to neutralize infectivity, and/or mediate antibody-complement, orantibody dependent cell cytotoxicity (ADCC) to provide protection to animmunized host. Such responses can be determined using standardimmunoassays and neutralization assays, well known in the art.

A vaccine composition which contains a selected antigen entrapped oradsorbed with a microparticle, along with a submicron oil-in-wateremulsion adjuvant, or a vaccine composition containing an antigenentrapped or adsorbed with a microparticle which is coadministered withthe subject submicron oil-in-water emulsion adjuvant, displays “enhancedimmunogenicity” when it possesses a greater capacity to elicit an immuneresponse than the immune response elicited by an equivalent amount ofthe microparticle/antigen without the submicron oil-in-water emulsionadjuvant. Thus, a vaccine composition may display “enhancedimmunogenicity” because the antigen is more strongly immunogenic orbecause a lower dose of antigen is necessary to achieve an immuneresponse in the subject to which it is administered. Such enhancedimmunogenicity can be determined by administering themicroparticle/antigen composition and submicron oil-in-water emulsion,and microparticle/antigen controls to animals and comparing antibodytiters against the two using standard assays such as radioimmunoassayand ELISAs, well known in the art.

The terms “effective amount” or “pharmaceutically effective amount” ofan agent, as provided herein, refer to a nontoxic but sufficient amountof the agent to provide the desired immunological response andcorresponding therapeutic effect. As will be pointed out below, theexact amount required will vary from subject to subject, depending onthe species, age, and general condition of the subject, the severity ofthe condition being treated, and the particular antigen of interest,mode of administration, and the like. An appropriate “effective” amountin any individual case may be determined by one of ordinary skill in theart using routine experimentation.

As used herein, “treatment” refers to any of (i) the prevention ofinfection or reinfection, as in a traditional vaccine, (ii) thereduction or elimination of symptoms, and (iii) the substantial orcomplete elimination of the pathogen in question. Treatment may beeffected prophylactically (prior to infection) or therapeutically(following infection).

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,i.e., the material may be administered to an individual along with themicroparticle adjuvant formulations without causing any undesirablebiological effects or interacting in a deleterious manner with any ofthe components of the composition in which it is contained.

By “physiological pH” or a “pH in the physiological range” is meant a pHin the range of approximately 7.2 to 8.0 inclusive, more typically inthe range of approximately 7.2 to 7.6 inclusive.

By “vertebrate subject” is meant any member of the subphylum cordata,including, without limitation, humans and other primates, includingnon-human primates such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, sheep, pigs, goats and horses;domestic mammals such as dogs and cats; laboratory animals includingrodents such as mice, rats and guinea pigs; birds, including domestic,wild and game birds such as chickens, turkeys and other gallinaceousbirds, ducks, geese, and the like. The term does not denote a particularage. Thus, both adult and newborn individuals are intended to becovered. The system described above is intended for use in any of theabove vertebrate species, since the immune systems of all of thesevertebrates operate similarly.

II. Modes of Carrying Out the Invention

The present invention is based on the discovery that the use ofmicroparticles with entrapped or adsorbed antigen, in combination withsubmicron oil-in-water emulsions, provide a vigorous immune response,even when the antigen is by itself weakly immunogenic. The submicronoil-in-water adjuvants of the present invention can be incorporated intovaccine compositions containing the desired microparticle/antigen, orcan be administered separately, either simultaneously with, just priorto, or subsequent to, a microparticle/antigen-containing composition.Furthermore, the formulations of the invention may be used to enhancethe activity of antigens produced in vivo, i.e., in conjunction with DNAimmunization.

Although the individual components of the vaccine compositions andmethods described herein were known, it was unexpected and surprisingthat such combinations would enhance the efficiency of antigens beyondlevels achieved when the components were used separately.

The method of the invention provides for cell-mediated immunity, and/orhumoral antibody responses. Thus, in addition to a conventional antibodyresponse, the system herein described can provide for, e.g., theassociation of the expressed antigens with class I MHC molecules suchthat an in vivo cellular immune response to the antigen of interest canbe mounted which stimulates the production of CTLs to allow for futurerecognition of the antigen. Furthermore, the methods may elicit anantigen-specific response by helper T-cells. Accordingly, the methods ofthe present invention will find use with any antigen for which cellularand/or humoral immune responses are desired, including antigens derivedfrom viral, bacterial, fungal and parasitic pathogens that may induceantibodies, T-cell helper epitopes and T-cell cytotoxic epitopes. Suchantigens include, but are not limited to, those encoded by human andanimal viruses and can correspond to either structural or non-structuralproteins.

The technique is particularly useful for immunization againstintracellular viruses and tumor cell antigens which normally elicit poorimmune responses. For example, the present invention will find use forstimulating an immune response against a wide variety of proteins fromthe herpesvirus family, including proteins derived from herpes simplexvirus (HSV) types 1 and 2, such as HSV-1 and HSV-2 glycoproteins gB, gDand gH; antigens derived from varicella zoster virus (VZV), Epstein-Barrvirus (EBV) and cytomegalovirus (CMV) including CMV gB and gH; andantigens derived from other human herpesviruses such as HHV6 and HHV7.(See, e.g. Chee et al., Cytomegaloviruses (J. K. McDougall, ed.,Springer-Verlag 1990) pp. 125-169, for a review of the protein codingcontent of cytomegalovirus; McGeoch et al., J. Gen. Virol. (1988)69:1531-1574, for a discussion of the various HSV-1 encoded proteins;U.S. Pat. No. 5,171,568 for a discussion of HSV-1 and HSV-2 gB and gDproteins and the genes encoding therefor; Baer et al., Nature (1984)310:207-211, for the identification of protein coding sequences in anEBV genome; and Davison and Scott, J. Gen. Virol. (1986) 67:1759-1816,for a review of VZV.)

Antigens from the hepatitis family of viruses, including hepatitis Avirus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the deltahepatitis virus (HDV), hepatitis E virus (HEV) and hepatitis G virus(HGV), can also be conveniently used in the techniques described herein.By way of example, the viral genomic sequence of HCV is known, as aremethods for obtaining the sequence. See, e.g., International PublicationNos. WO 89/04669; WO 90/11089; and WO 90/14436. The HCV genome encodesseveral viral proteins, including E1 (also known as E) and E2 (alsoknown as E2/NSI) and an N-terminal nucleocapsid protein (termed “core”)(see, Houghton et al., Hepatology (1991) 14:381-388, for a discussion ofHCV proteins, including E1 and E2). Each of these proteins, as well asantigenic fragments thereof, will find use in the present methods.Similarly, the sequence for the δ-antigen from HDV is known (see, e.g.,U.S. Pat. No. 5,378,814) and this antigen can also be conveniently usedin the present methods. Additionally, antigens derived from HBV, such asthe core antigen, the surface antigen, sAg, as well as the presurfacesequences, pre-S1 and pre-S2 (formerly called pre-S), as well ascombinations of the above, such as sAg/pre-S1, sAg/pre-S2,sAg/pre-S1/pre-S2, and pre-S1/pre-S2, will find use herein. See, e.g.,“HBV Vaccines—from the laboratory to license: a case study” in Mackett,M. and Williamson, J. D., Human Vaccines and Vaccination, pp. 159-176,for a discussion of HBV structure; and U.S. Pat. Nos. 4,722,840,5,098,704, 5,324,513, incorporated herein by reference in theirentireties; Beames et al., J. Virol. (1995) 69:6833-6838, Birnbaum etal., J. Virol. (1990) 64:3319-3330; and Zhou et al., J. Virol. (1991)65:5457-5464.

Antigens derived from other viruses will also find use in the claimedmethods, such as without limitation, proteins from members of thefamilies Picornaviridae (e.g., polioviruses, etc.); Caliciviridae;Togaviridae (e.g., rubella virus, dengue virus, etc.); Flaviviridae;Coronaviridae; Reoviridae; Birnaviridae; Rhabodoviridae (e.g., rabiesvirus, etc.); Filoviridae; Paramyxoviridae (e.g., mumps virus, measlesvirus, respiratory syncytial virus, etc.); Orthomyxoviridae (e.g.,influenza virus types A, B and C, etc.); Bunyaviridae; Arenaviridae;Retroviradae (e.g., HTLV-I; HTLV-II; HIV-1 (also known as HTLV-III, LAV,ARV, hTLR, etc.)), including but not limited to antigens from theisolates HIV_(IIIb), HIV_(SF2), HIV_(LAV), HIV_(LAI), HIV_(MN));HIV-1_(CM235), HIV-1_(US4); HIV-2; simian immunodeficiency virus (SIV)among others. Additionally, antigens may also be derived from humanpapillomavirus (HPV) and the tick-borne encephalitis viruses. See, e.g.Virology, 3rd Edition (W. K. Joklik ed. 1988); Fundamental Virology, 2ndEdition (B. N. Fields and D. M. Knipe, eds. 1991), for a description ofthese and other viruses.

More particularly, the gp120 envelope proteins from any of the above HIVisolates, including members of the various genetic subtypes of HIV, areknown and reported (see, e.g., Myers et al., Los Alamos Database, LosAlamos National Laboratory, Los Alamos, N. Mex. (1992); Myers et al.,Human Retroviruses and Aids, 1990, Los Alamos, N. Mex.: Los AlamosNational Laboratory; and Modrow et al., J. Virol. (1987) 61:570-578, fora comparison of the envelope sequences of a variety of HIV isolates) andantigens derived from any of these isolates will find use in the presentmethods. Furthermore, the invention is equally applicable to otherimmunogenic proteins derived from any of the various HIV isolates,including any of the various envelope proteins such as gp160 and gp41,gag antigens such as p24gag and p55gag, as well as proteins derived fromthe pol region.

As explained above, influenza virus is another example of a virus forwhich the present invention will be particularly useful. Specifically,the envelope glycoproteins HA and NA of influenza A are of particularinterest for generating an immune response. Numerous HA subtypes ofinfluenza A have been identified (Kawaoka et al., Virology (1990)179:759-767; Webster et al., “Antigenic variation among type A influenzaviruses,” p. 127-168. In: P. Palese and D. W. Kingsbury (ed.), Geneticsof influenza viruses. Springer-Verlag, New York). Thus, proteins derivedfrom any of these isolates can also be used in the immunizationtechniques described herein.

The methods described herein will also find use with numerous bacterialantigens, such as those derived from organisms that cause diphtheria,cholera, tuberculosis, tetanus, pertussis, meningitis, and otherpathogenic states, including, without limitation, Meningococcus A, B andC, Hemophilus influenza type B (HIB), and Helicobacter pylori. Examplesof parasitic antigens include those derived from organisms causingmalaria and Lyme disease.

Furthermore, the methods described herein provide a means for treating avariety of malignant cancers. For example, the system of the presentinvention can be used to mount both humoral and cell-mediated immuneresponses to particular proteins specific to the cancer in question,such as an activated oncogene, a fetal antigen, or an activation marker.Such tumor antigens include any of the various MAGEs (melanomaassociated antigen E), including MAGE 1, 2, 3, 4, etc. (Boon, T.Scientific American (March 1993):82-89); any of the various tyrosinases;MART 1 (melanoma antigen recognized by T cells), mutant ras; mutant p53;p97 melanoma antigen; CEA (carcinoembryonic antigen), among others.

It is readily apparent that the subject invention can be used to preventor treat a wide variety of diseases.

The selected antigen is entrapped in, or adsorbed to, a microparticlefor subsequent delivery. Biodegradable polymers for manufacturingmicroparticles useful in the present invention are readily commerciallyavailable from, e.g., Boehringer Ingelheim, Germany and BirminghamPolymers, Inc., Birmingham, Ala. For example, useful polymers forforming the microparticles herein include those derived frompolyhydroxybutyric acid; polycaprolactone; polyorthoester;polyanhydride; as well as a poly(α-hydroxy acid), such aspoly(L-lactide), poly(D,L-lactide) (both known as “PLA” herein),poly(hydoxybutyrate), copolymers of D,L-lactide and glycolide, such aspoly(D,L-lactide-co-glycolide) (designated as “PLG” or “PLGA” herein) ora copolymer of D,L-lactide and caprolactone. Particularly preferredpolymers for use herein are PLA and PLG polymers. These polymers areavailable in a variety of molecular weights, and the appropriatemolecular weight for a given antigen is readily determined by one ofskill in the art. Thus, e.g., for PLA, a suitable molecular weight willbe on the order of about 2000 to 250,000. For PLG, suitable molecularweights will generally range from about 10,000 to about 200,000,preferably about 15,000 to about 150,000, and most preferably about50,000 to about 100,000.

If a copolymer such as PLG is used to form the microparticles, a varietyof lactide:glycolide ratios will find use herein and the ratio islargely a matter of choice, depending in part on the coadministeredantigen and the rate of degradation desired. For example, a 50:50 PLGpolymer, containing 50% D,L-lactide and 50% glycolide, will provide afast resorbing copolymer while 75:25 PLG degrades more slowly, and 85:15and 90:10, even more slowly, due to the increased lactide component. Itis readily apparent that a suitable ratio of lactide:glycolide is easilydetermined by one of skill in the art based on the nature of the antigenand disorder in question. Moreover, mixtures of microparticles withvarying lactide:glycolide ratios will find use in the formulations inorder to achieve the desired release kinetics for a given antigen and toprovide for both a primary and secondary immune response. Degradationrate of the microparticles of the present invention can also becontrolled by such factors as polymer molecular weight and polymercrystallinity. PLG copolymers with varying lactide:glycolide ratios andmolecular weights are readily available commercially from a number ofsources including from Boehringer Ingelheim, Germany and BirminghamPolymers, Inc., Birmingham, Ala. These polymers can also be synthesizedby simple polycondensation of the lactic acid component using techniqueswell known in the art, such as described in Tabata et al., J. Biomed.Mater. Res. (1988) 22:837-858.

The antigen/microparticles are prepared using any of several methodswell known in the art. For example, double emulsion/solvent evaporationtechniques, such as described in U.S. Pat. No. 3,523,907 and Ogawa etal., Chem. Pharm. Bull. (1988) 36:1095-1103, can be used herein to formthe microparticles. These techniques involve the formation of a primaryemulsion consisting of droplets of polymer solution containing theantigen (if antigen is to be entrapped in the microparticle), which issubsequently mixed with a continuous aqueous phase containing a particlestabilizer/surfactant.

More particularly, a water-in-oil-in-water (w/o/w) solvent evaporationsystem can be used to form the microparticles, as described by O'Haganet al., Vaccine (1993) 11:965-969 and Jeffery et al., Pharm. Res. (1993)10:362. In this technique, the particular polymer is combined with anorganic solvent, such as ethyl acetate, dimethylchloride (also calledmethylene chloride and dichloromethane), acetonitrile, acetone,chloroform, and the like. The polymer will be provided in about a 2-15%,more preferably about a 4-10% and most preferably, a 6% solution, inorganic solvent. An approximately equal amount of an antigen solution,e.g., in water, is added and the polymer/antigen solution emulsifiedusing e.g, an homogenizer. The emulsion is then combined with a largervolume of an aqueous solution of an emulsion stabilizer such aspolyvinyl alcohol (PVA) or polyvinyl pyrrolidone. The emulsionstabilizer is typically provided in about a 2-15% solution, moretypically about a 4-10% solution. The mixture is then homogenized toproduce a stable w/o/w double emulsion. Organic solvents are thenevaporated.

The formulation parameters can be manipulated to allow the preparationof small (<5 μm) and large (>30 μm) microparticles. See, e.g., Jefferyet al., Pharm. Res. (1993) 10:362-368; McGee et al., J. Microencap.(1996). For example, reduced agitation results in larger microparticles,as does an increase in internal phase volume. Small particles areproduced by low aqueous phase volumes with high concentrations of PVA.

Microparticles can also be formed using spray-drying drying andcoacervation as described in, e.g., Thomasin et al., J. ControlledRelease (1996) 41:131; U.S. Pat. No. 2,800,457; Masters, K. (1976) SprayDrying 2nd Ed. Wiley, N.Y.; air-suspension coating techniques, such aspan coating and Wurster coating, as described by Hall et al., (1980) The“Wurster Process” in Controlled Release Technologies: Methods, Theory,and Applications (A. F. Kydonieus, ed.), Vol. 2, pp. 133-154 CRC Press,Boca Raton, Fla. and Deasy, P. B., Crit. Rev. Ther. Drug Carrier Syst.(1988) S (2):99-139; and ionic gelation as described by, e.g., Lim etal., Science (1980) 210:908-910.

The above techniques are also applicable to the production ofmicroparticles with adsorbed antigens. In this embodiment,microparticles are formed as described above, however, antigens aremixed with the microparticles following formation.

Particle size can be determined by, e.g., laser light scattering, usingfor example, a spectrometer incorporating a helium-neon laser.Generally, particle size is determined at room temperature and involvesmultiple analyses of the sample in question (e.g., 5-10 times) to yieldan average value for the particle diameter. Particle size is alsoreadily determined using scanning electron microscopy (SEM).

Prior to use of the microparticles, antigen content is generallydetermined so that an appropriate amount of the microparticles may bedelivered to the subject in order to elicit an adequate immune response.Antigen content of the microparticles can be determined according tomethods known in the art, such as by disrupting the microparticles andextracting the entrapped antigen. For example, microparticles can bedissolved in dimethylchloride and the protein extracted into distilledwater, as described in, e.g., Cohen et al., Pharm. Res. (1991) 8:713;Eldridge et al., Infect. Immun. (1991) 59:2978; and Eldridge et al., J.Controlled Release (1990)11:205. Alternatively, microparticles can bedispersed in 0.1 M NaOH containing 5% (w/v) SDS. The sample is agitated,centrifuged and the supernatant assayed for the antigen of interestusing an appropriate assay. See, e.g., O'Hagan et al., Int. J. Pharm.(1994) 103:37-45.

As explained above, a submicron oil-in-water emulsion formulation willalso be administered to the vertebrate subject, either prior to,concurrent with, or subsequent to, delivery of theantigen/microparticle.

Submicron oil-in water emulsions for use herein include nontoxic,metabolizable oils and commercial emulsifiers. Examples of nontoxic,metabolizable oils include, without limitation, vegetable oils, fishoils, animal oils or synthetically prepared oils. Fish oils, such as codliver oil, shark liver oils and whale oils, are preferred, withsqualene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene,found in shark liver oil, particularly preferred. The oil component willbe present in an amount of from about 0.5% to about 20% by volume,preferably in an amount up to about 15%, more preferably in an amount offrom about 1% to about 12% and most preferably from 1% to about 4% oil.

The aqueous portion of the adjuvant can be buffered saline orunadulterated water. Since the compositions are intended for parenteraladministration, it is preferable to make up the final solutions so thatthe tonicity, i.e., osmolality, is essentially the same as normalphysiological fluids, in order to prevent post-administration swellingor rapid absorption of the composition due to differential ionconcentrations between the composition and physiological fluids. Ifsaline is used rather than water, it is preferable to buffer the salinein order to maintain a pH compatible with normal physiologicalconditions. Also, in certain instances, it may be necessary to maintainthe pH at a particular level in order to insure the stability of certaincomposition components. Thus, the pH of the compositions will generallybe pH 6-8 and pH can be maintained using any physiologically acceptablebuffer, such as phosphate, acetate, tris, bicarbonate or carbonatebuffers, or the like. The quantity of the aqueous agent present willgenerally be the amount necessary to bring the composition to thedesired final volume.

Emulsifying agents suitable for use in the oil-in-water formulationsinclude, without limitation, sorbitan-based non-ionic surfactants suchas those commercially available under the name of SPAN®or ARLACEL®;polyoxyethylene sorbitan monoesters and polyoxyethylene sorbitantriesters, commercially known by the name TWEEN®; polyoxyethylene fattyacids available under the name MYRJ®; polyoxyethylene fatty acid ethersderived from lauryl, acetyl, stearyl and oleyl alcohols, such as thoseknown by the name of BRIJ®; and the like. These substances are readilyavailable from a number of commercial sources, including ICI America'sInc., Wilmington, Del. These emulsifying agents may be used alone or incombination. The emulsifying agent will usually be present in an amountof 0.02% to about 2.5% by weight (w/w), preferably 0.05% to about 1%,and most preferably 0.01% to about 0.5. The amount present willgenerally be about 20-30% of the weight of the oil used.

The emulsions can also contain other immunostimulating agents, such asmuramyl peptides, including, but not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc. Immunostimulating bacterial cell wall components, such asmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), may also be present.

For a description of various suitable submicron oil-in-water emulsionformulations for use with the present invention, see, e.g.,International Publication No. WO 90/14837; Remington: The Science andPractice of Pharmacy, Mack Publishing Company, Easton, Pa., 19thedition, 1995; Van Nest et al., “Advanced adjuvant formulations for usewith recombinant subunit vaccines,” In Vaccines 92, Modern Approaches toNew Vaccines (Brown et al., ed.) Cold Spring Harbor Laboratory Press,pp. 57-62 (1992); and Ott et al., “MF59—Design and Evaluation of a Safeand Potent Adjuvant for Human Vaccines” in Vaccine Design: The Subunitand Adjuvant Approach (Powell, M. F. and Newman, M. J. eds.) PlenumPress, New York (1995) pp. 277-296.

In order to produce submicron particles, i.e., particles less than 1micron in diameter and in the nanometer size range, a number oftechniques can be used. For example, commercial emulsifiers can be usedthat operate by the principle of high shear forces developed by forcingfluids through small apertures under high pressure. Examples ofcommercial emulsifiers include, without limitation, Model 110Ymicrofluidizer (Microfluidics, Newton, Mass.), Gaulin Model 30CD(Gaulin, Inc., Everett, Mass.), and Rainnie Minilab Type 8.30H (MiroAtomizer Food and Dairy, Inc., Hudson, Wis.). The appropriate pressurefor use with an individual emulsifier is readily determined by one ofskill in the art. For example, when the Model 110Y microfluidizer isused, operation at 5000 to 30,000 psi produces oil droplets withdiameters of about 100 to 750 nm.

The size of the oil droplets can be varied by changing the ratio ofdetergent to oil (increasing the ratio decreases droplet size),operating pressure (increasing operating pressure reduces droplet size),temperature (increasing temperature decreases droplet size), and addingan amphipathic immunostimulating agent (adding such agents decreasesdroplet size). Actual droplet size will vary with the particulardetergent, oil and immunostimulating agent (if any) and with theparticular operating conditions selected. Droplet size can be verifiedby use of sizing instruments, such as the commercial Sub-Micron ParticleAnalyzer (Model N4MD) manufactured by the Coulter Corporation, and theparameters can be varied using the guidelines set forth above untilsubstantially all droplets are less than 1 micron in diameter,preferably less than about 0.8 microns in diameter, and most preferablyless than about 0.5 microns in diameter. By substantially all is meantat least about 80% (by number), preferably at least about 90%, morepreferably at least about 95%, and most preferably at least about 98%.The particle size distribution is typically Gaussian, so that theaverage diameter is smaller than the stated limits.

Particularly preferred submicron oil-in-water emulsions for use hereinare squalene/water emulsions optionally containing varying amounts ofMTP-PE, such as the submicron oil-in-water emulsion known as “MF59”(International Publication No. WO 90/14837; Ott et al., “M F59—Designand Evaluation of a Safe and Potent Adjuvant for Human Vaccines” inVaccine Design: The Subunit and Adjuvant Approach (Powell, M. F. andNewman, M. J. eds.) Plenum Press, New York, 1995, pp. 277-296). MF59contains 4-5% w/v Squalene (e.g., 4.3%), 0.25-0.5% w/v TWEEN 80®, and0.5% w/v SPAN 85® and optionally contains various amounts of MTP-PE,formulated into submicron particles using a microfluidizer such as Model11 OY microfluidizer (Microfluidics, Newton, Mass.). For example, MTP-PEmay be present in an amount of about 0-500 μg/dose, more preferably0-250 μg/dose and most preferably, 0-100 μg/dose. MF59-0, therefore,refers to the above submicron oil-in-water emulsion lacking MTP-PE,while MF59-100 contains 100 μg MTP-PE per dose. MF69, another submicronoil-in-water emulsion for use herein, contains 4.3% w/v squalene, 0.25%w/v TWEEN 80®, and 0.75% w/v SPAN 85® an optionally MTP-PE. Yet anothersubmicron oil-in-water emulsion is SAF, containing 10% squalene, 0.4%TWEEN 80®, 5% pluronic-blocked polymer L121, and thr-MDP, alsomicrofluidized into a submicron emulsion.

Once the submicron oil-in-water emulsion is formulated it can beadministered to the vertebrate subject, either prior to, concurrentwith, or subsequent to, delivery of the microparticle. If administeredprior to immunization with the microparticle, the adjuvant formulationscan be administered as early as 5-10 days prior to immunization,preferably 3-5 days prior to immunization and most preferably 1-3 or 2days prior to immunization with the antigens of interest. Ifadministered separately, the submicron oil-in-water formulation can bedelivered either to the same site of delivery as the microparticlecompositions or to a different delivery site.

If simultaneous delivery is desired, the submicron oil-in-waterformulation can be included with the microparticle compositions.Generally, the microparticles and submicron oil-in-water emulsion can becombined by simple mixing, stirring, or shaking. Other techniques, suchas passing a mixture of the two components rapidly through a smallopening (such as a hypodermic needle) can also be used to provide thevaccine compositions.

If combined, the various components of the composition can be present ina wide range of ratios. For example, the microparticle and emulsioncomponents are typically used in a volume ratio of 1:50 to 50:1,preferably 1:10 to 10:1, more preferably from about 1:3 to 3:1, and mostpreferably about 1:1. However, other ratios may be more appropriate forspecific purposes, such as when a particular antigen is both difficultto incorporate into a microparticle and has a low immungenicity, inwhich case a higher relative amount of the antigen component isrequired.

Once formulated, the compositions of the invention are administeredparenterally, generally by injection. The compositions can be injectedeither subcutaneously, intraperitoneally, intravenously orintramuscularly. Dosage treatment may be a single dose schedule or amultiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may be with 1-10 separate doses, followedby other doses given at subsequent time intervals, chosen to maintainand/or reinforce the immune response, for example at 1-4 months for asecond dose, and if needed, a subsequent dose(s) after several months.The boost may be with a microparticle/submicron oil-water-emulsion givenfor the primary immune response, or may be with a different formulationthat contains the antigen. The dosage regimen will also, at least inpart, be determined by the need of the subject and be dependent on thejudgment of the practitioner. Furthermore, if prevention of disease isdesired, the vaccines are generally administered prior to primaryinfection with the pathogen of interest. If treatment is desired, e.g.,the reduction of symptoms or recurrences, the vaccines are generallyadministered subsequent to primary infection.

The compositions will generally include one or more “pharmaceuticallyacceptable excipients or vehicles” such as water, saline, glycerol,polyethyleneglycol, hyaluronic acid, ethanol, etc. Additionally,auxiliary substances, such as wetting or emulsifying agents, pHbuffering substances, and the like, may be present in such vehicles.

The compositions will comprise a “therapeutically effective amount” ofthe antigen of interest. That is, an amount of antigen will be includedin the compositions which, when in combination with the submicron-oil-inwater emulsion, will cause the subject to produce a sufficientimmunological response in order to prevent, reduce or eliminatesymptoms. The exact amount necessary will vary, depending on the subjectbeing treated; the age and general condition of the subject to betreated; the capacity of the subject's immune system to synthesizeantibodies; the degree of protection desired; the severity of thecondition being treated; the particular antigen selected and its mode ofadministration, among other factors. An appropriate effective amount canbe readily determined by one of skill in the art. Thus, a“therapeutically effective amount” will fall in a relatively broad rangethat can be determined through routine trials. For example, for purposesof the present invention, an effective dose will typically range fromabout 1 μg to about 100 mg, more preferably from about 10 μg to about 1mg, and most preferably about 50 μg to about 500 μg of the antigendelivered per dose.

III. Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

EXAMPLE 1 Preparation of p24 Antigen-Entrapped Microparticles

Materials used to formulate the microparticles were as follows:

(1) poly(D,L-lactide-co-glycolide), 50:50 mol ratio lactide toglycolide, MW average=70-100 kDa (50:50 PLGA high viscosity polymer)(Medisorb Technologies International, Cincinnati, Ohio);

(2) 8% polyvinyl alcohol (PVA) 3-83 (Mowiol, Frankfurt, Germany) inwater saturated 10% with ethyl acetate by adding 6 ml of the latter andstirring in a screw cap glass bottle for 10 minutes; and

(3) p24gag/sf2, in 30 mM Tris, pH 7.5, at a concentration of 6.1 mg ofantigen/ml with 20:1 sucrose:protein.

P24gag microparticles were prepared by a solvent extraction technique asfollows. To make the microparticles, 2.58 g of the polymer solution wassonicated with 0.8 ml of the antigen solution for 30 seconds. Theprimary emulsion was homogenized with 60 grams of the saturated PVAsolution using a benchtop homogenizer with a 20 mm probe at 10 K rpm for1 minute. The resulting emulsion was immediately added to 2.5 L ofwater, stirred for two hours for solvent extraction, filtered through a38 μmesh, washed by centrifugation 3 times, and a portion sonicated forone minute in a water bath sonicator, then sized by laser diffractionmeasurement. The mean diameter size of the microparticles was 10 μm. Themicrospheres were lyophilized and stored at −20° C.

EXAMPLE 2 Immungenicity of p24gag/sf2 Antigen-Entrapped Microparticleswith MF59

10 Baboons were divided into two groups (five baboons per group) andadministered the formulations specified in Table 1. For Group 1, equalparts of the adjuvant MF59-0, and p24gag/sf2 (in a citrate/Tris buffer)were combined to yield a total of 0.7 ml. The composition was gentlymixed and 500 μl (to yield 100 μg p24gag/sf2/dose) of vaccine wasinjected intramuscularly (IM) in the thigh muscle. For Group 2, 1.5 mlof 2× phosphate buffered saline (PBS) was added to 46.8 mg of thePLG-entrapped p24gag/sf2 formulation produced in Example 1. The materialwas vortexed for about 30 seconds until all beads were in suspension.1.5 ml of MF59-0 was added to the resuspended beads to yield a total of3 ml. The composition was gently mixed and 500 μl (to yield 100 μgp24gag/sf2/dose) of vaccine was injected IM in the thigh muscle.

Both groups of animals were boosted twice at 4 week intervals followingthe initial injection, with 500 μl of the vaccine composition. Two weeksfollowing the second boost (10 weeks after the initial immunization)serum was collected and IgG titers evaluated by a standard ELISA,essentially as described below.

As shown in Table 1, entrapped p24gag/sf2+MF59 elicited a significantlygreater antibody response than nonentrapped p24gag/sf2+MF59.

TABLE 1 Mean IgG Group/Formulation titers Group 1 p24gag/sf2 100 μg +MF59-0 19,976 Group 2 p24gag/sf2 100 μg in PLG 85,725 Microparticles +MF59-0

EXAMPLE 3 Preparation of qp120/sf2 Antigen-Entrapped Microparticles

Materials used to formulate the microparticles were as follows:

(1) 3.0 g of the polymer poly(D,L-lactide-co-glycolide) composed of a50:50 mol ratio of lactide to glycolide with a molecular weight averageof 80 Kdal, (Boehringer Ingelheim Resomer RG505), was dissolved in 50 mlof dichloromethane (DCM, HPLC grade, obtained from Aldrich);

(2) 16 g polyvinyl alcohol (13-23 Kdal molecular weight average, ICNBiomedicals, Aurora, Ohio) was dissolved in 200 ml deionized water; and

(3) HIV gp120sf2 antigen (Chiron, clinical grade) was used, at aconcentration of 7.2 mg antigen/ml in 30 mM sodium citrate, pH 6.0,buffer.

Microparticles were prepared as follows. 1.67 ml of the HIV gp120sf2antigen were added to 16.7 ml of the poly(D,L-lactide-co-glycolide)solution in a 30 ml glass heavy-walled test tube. The solution washomogenized 3 minutes at 23,000 RPM using a small, hand-held homogenizerequipped with 10 mm diameter generator. The homogenate was then slowlypoured into 66.8 ml of the polyvinyl alcohol solution in a 150 ml glassbeaker while homogenizing at 12,000 RPM using a bench scale homogenizerequipped with a 20 mm diameter generator for a total homogenization timeof 3 minutes. The beaker containing the resulting double emulsion wasequipped with a small magnetic stir bar. This was then allowed to sitovernight at ambient temperature under moderate (approximately 1000 RPM)stirring rate to evaporate the DCM solvent. The resulting microparticlesprepared in this way were washed to remove excess PVA and un-entrappedantigen. Washing was accomplished by repeatedly (3 times total) dilutingthe microparticle preparation in approximately 450 ml deionized water,centrifuging to pellet microparticles, decanting off supernatant, andresuspending the microparticles in approximately 30 ml deionized water.After the final resuspension step, the microparticles were lyophilizedand stored at −20° C.

Small samples (10-30 mg) of the lyophilized microparticles were utilizedto measure particle size distribution and antigen content. The sizedistribution of the microparticles thus prepared was measured by dynamiclaser light scatter using a Malvern Mastersizer instrument anddetermined to have a median size of 0.6 μm. The antigen content (% load)was measured by dissolving samples of the microparticles in a 0.1 Msodium hydroxide, 1% sodium dodecyl sulfate solution, then measuringprotein content using a standard bicinchoninic acid (BCA) assay (Pierce,Rockford, Ill.). The % load of the microparticles was measured in thismanner and determined to contain 0.7% protein by weight.

EXAMPLE 4 Immungenicity of qp120/sf2 Antigen-Entrapped Microparticleswith MF59 in Baboons

A similar experiment was run as described in Example 2, using gp102/sf2in place of p24gag/sf2. In particular, gp120/sf2 was combined withMF59-0 and 50 μg administered to Group 1 baboons, as described above.Additionally, the PLG-entrapped gp120/sf2 from Example 1 was combinedwith MF59-0 as described and 50 μg administered to the Group 2 animals.

Both groups of animals were boosted at 4 weeks following the initialinjection, with 500 μl of the vaccine composition. Serum samples werecollected four weeks after the initial dose (4wp1), as well as fourweeks following the second dose (4wp2) and 8 weeks following the seconddose (8wp2) and IgG titers evaluated by ELISA as follows. 96-well ELISAplates (Nunc U96, cat#449824) were coated with 100 μl per well of 2μg/ml gp120/sf2 antigen in 50 mM sodium borate buffer, pH 9.0. Theplates were incubated overnight at 4° C. Baboon serum samples, initiallydiluted 1:50 to 1:1000 in 100 mM sodium phosphate, 1 mM EDTA, 0.5 Msodium chloride buffer, pH 7.5 (dilution solution), were seriallydiluted with dilution solution 1:2 from top to bottom of the ELISA plate(one column per serum sample) such that samples were diluted by a factorof 1-, 2-, 4-, 8-, 16-, 32-, 64- and 128-fold greater than the initialdilution, with a final volume of 100 μl sample per well. A columncontaining dilution solution only (blank), and a standard serum(standard) were included on each plate for comparison purposes. ELISAplates were incubated 1 hour at 37° C. After washing plates extensivelywith 0.05% Triton-X100 solution, 100 μl per well of a 1:5000 dilutedGoat anti-Monkey IgG-HRP conjugate solution (Organon Teknike Corp., WestChester, Pa., cat#55432) was added. Plates were incubated 1 hour at 37°C. Plates were again washed extensively with 0.05% Triton-X100. 100 μlTMB peroxide developer solution (Kirkegaard & Perry labs, Gaithersburg,Md.) were added to each well. Color reaction was allowed to develop forapproximately 3 minutes before stopping by adding 50 μl per well 2 M HCl. Plates were read using an ELISA reader at 450 nm. Resulting OD valuesfor each plate were subtracted from baseline OD using average valuesfrom a blank column. Titers for each serum sample were expressed as thedilution required to achieve an OD of 0.5 as determined by fittingresulting data to a log-logit function.

As shown in Table 2, entrapped gp120/sf2+MF59 elicited a greaterantibody response than nonentrapped gp120/sf2+MF59 in all groups withthe response seen at four weeks after the first dose being significantlyhigher.

TABLE 2 Mean IgG Mean IgG Mean IgG titers titers titersGroup/Formulation 4wp1 4wp2 8wp2 Group 1 gp120/sf2 50 μg + MF59-0 103297 1118 Group 2 gp120/sf2 50 μg entrapped in 637 5120 1733 PLGMicroparticles + MF59-0

EXAMPLE 5 Immungenicity of gp120/sf2 Antigen-Entrapped Microparticleswith MF59 in Mice

The ability of HIV gp120 to stimulate an immune response when entrappedor adsorbed to PLG microparticles and coadministered with MF59 was alsotested in mice as follows. Balb/C mice, 6-7 weeks in age, were dividedinto four groups and administered intramuscularly 50 μl of a vaccinecomposition containing 10 μg of HIV gp120, and adjuvant as specified inTable 3. The various compositions were prepared as described in Example4 above.

TABLE 3 Animal Adjuvant Antigen Volume per Injection Group numbers NameDose Name Dose Site Animal Route 1 (10) PBS HIV 10 μg 50 μl 50 μl IMgP120 (soluble) 2 (30) MF59-0 25 μl HIV 10 μg 50 μl 50 μl IM gP120(soluble) 3 (30) PLG/ 1.3 HIV 10 μg 50 μl 50 μl IM gp120 mg gP120(entrapped) 4 (30) PLG/gp120 1.3 HIV 10 μg 50 μl 50 μl IM in MF59-0 mg25 gP120 μl (entrapped)

Animals were boosted at 4 and 8 weeks following the initial injection.Serum was collected at 2, 6 and 10 weeks following injection and IgGtiters evaluated by a standard ELISA, as described in Example 4.

The results are shown in Table 4 and FIG. 1. In all cases, IgG titerswere higher in the group administered PLG-entrapped gp120+MF59 than IgGtiters in the other groups, and significantly higher than the groupadministered MF59 alone. At 10 weeks following injection, IgG titerswere significantly higher in the group adminstered PLG-entrappedgp120+MF59 as compared to all other groups.

TABLE 4 Total IgG Formulation 2 weeks 6 weeks 10 weeks gp120 9 9 19gp120 + MF59 9 65 851 PLG/gp120 54 40728 62167 PLG/gp120 + MF59 82 70672113172

EXAMPLE 6 Immungenicity of HCV E2 Antigen-Entrapped Microparticles withMF59 in Mice

The ability of the hepatitis C virus (HCV) E2 antigen to stimulate animmune response when entrapped or adsorbed to PLG microparticles andcoadministered with MF59 was tested as follows. Mice were divided intosix groups and administered intramuscularly 50 μl of a vaccinecomposition containing 5 μg of HCV E2 antigen and adjuvant as specifiedin Table 5. The compositions were prepared essentially as describedabove.

Animals were boosted at 4 and 8 weeks following the initial injection.Serum was collected at 2, 6, 10 and 12 weeks following injection and IgGtiters evaluated by a standard ELISA, essentially as described above.

As shown in Table 6, antibody titers for HCV E2, either adsorbed orentrapped in PLG microparticles, and coadministered with MF59, werehigher than those seen when PLG or MF59 were administered alone.

TABLE 5 Adjuvant E2 Group # Name Dose Dose 1 MF59  50 μl — 2 PLG mixed500 μg 5 μg 3 PLG adsorbed 500 μg 5 μg 4 PLG entrapped 500 μg 5 μg 5 PLGadsorbed + MF59 500 μg 5 μg 6 PLG entrapped + MF59 500 μg 5 μg

TABLE 6 PLG PLG PLG PLG ads. + entr. + Weeks MF59 PLG adsorbed entrappedMF59 MF59 0 0.43 0.39 0.42 0.5 0.47 0.52 2 0.37 0.21 0.19 0.28 2.26 0.546 31.48 4.71 5.67 49.96 98.77 175.69 10 155.04 6.74 31.35 176 418 425 12141 1.13 20.33 21.66 123 188

Accordingly, the use of submicron oil-in-water emulsions withantigen-entrapped and -adsorbed microparticles is disclosed. Althoughpreferred embodiments of the subject invention have been described insome detail, it is understood that obvious variations can be madewithout departing from the spirit and the scope of the invention asdefined by the appended claims.

1. A method of inducing an immune response which comprises administeringto a vertebrate subject (a) a submicron oil-in-water emulsionimmunological adjuvant, and (b) a therapeutically effective amount of aselected antigen entrapped in, or adsorbed to, a biodegradablemicroparticle, wherein the submicron oil-in-water emulsion comprises 1%to 12% by volume of a non-toxic metabolizable oil and 0.02% to 2.5%(w/v) of emulsifying agent.
 2. The method of claim 1, wherein themicroparticle is formed from a poly(α-hydroxy acid) selected from thegroup consisting of poly(L-lactide), poly(D,L-Iactide) andpoly(D,L-lactide-co-glycolide).
 3. The method of claim 2, wherein themicroparticle is formed from poly(D,L-lactide-co-glycolide.
 4. Themethod of claim 1, wherein the submicron oil-in-water emulsion comprises4-5% w/v squalene, 0.25-0.5% w/v polyoxyethylene sorbitan monooleate,and 0.5% w/v sorbitan trioleate, and optionally,N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine.5. The method of claim 1, wherein the selected antigen is a viralantigen.
 6. The method of claim 5, wherein the selected antigen is gp120.
 7. The method of claim 5, wherein the selected antigen is p24gag.8. The method of claim 5, wherein the selected antigen is hepatitis Cvirus E2.
 9. The method of claim 1, wherein the selected antigen isentrapped in the microp article.
 10. The method of claim 1, wherein theselected antigen is adsorbed to the microparticle.
 11. The method ofclaim 1, wherein the submicron oil-in-water emulsion is administeredprior to the microparticle.
 12. The method of claim 1, wherein thesubmicron oil-in-water emulsion is administered subsequent to themicroparticle.
 13. The method of claim 1, wherein the submicronoil-in-water emulsion is administered substantially concurrently withthe microparticle.
 14. The method of claim 1, wherein the submicronoil-in-water emulsion further comprisesN-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine.15. the method of claim 4, wherein the submicron oil-in-water emulsionfurther comprisesN-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine.